DSOF: A Rapid Method to Determine the Abundance of Microalgae and Methanotrophic Bacteria in Coculture Using a Combination of Differential Sedimentation, Optical Density, and Fluorescence

Cocultivation of microalgae and aerobic methanotrophs represents an emerging biotechnology platform to produce high-protein biomass, yet quantifying individual species in mixed cultures remains challenging. Here, we present a rapid, low-cost method—differential sedimentation, optical density, and fluorescence (DSOF)—to determine the abundance of coculture members. DSOF exploits differences in cell size and pigment autofluorescence between the thermoacidophilic microalga and methanotrophic species Galdieria sp. RTK37.1 and Methylacidiphilum sp. RTK17.1, respectively, to selectively sediment algal cells and estimate population contributions via OD600 and phycocyanin fluorescence. Evaluation with model suspensions across a wide cell density range (0 ≤ [Galdieria]: ≤ 3.23 A.U., and 0 ≤ [Methylacidiphilum] ≤ 1.54 A.U.) showed strong agreement with known values, with most absolute errors < 0.1 A.U. and relative errors < 10% at moderate biomass levels. Application to live batch cocultures under microalga or methanotroph growth-suppressed conditions, and during simultaneous growth, demonstrated accurate tracking of population dynamics and revealed enhanced methanotroph growth in the presence of oxygenic microalgae. While DSOF accuracy decreases at very concentrated biomass (>2.0 A.U. for Galdieria) or under nitrogen-limiting conditions, the model provides a practical, scalable alternative to more complex, invasive or expensive techniques, enabling near real-time monitoring of microalgae–methanotroph cocultures.